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EP2663323A1 - Procédés de ciblage du mir-128 en vue de la régulation du métabolisme du cholestérol/des lipides - Google Patents

Procédés de ciblage du mir-128 en vue de la régulation du métabolisme du cholestérol/des lipides

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Publication number
EP2663323A1
EP2663323A1 EP12734471.1A EP12734471A EP2663323A1 EP 2663323 A1 EP2663323 A1 EP 2663323A1 EP 12734471 A EP12734471 A EP 12734471A EP 2663323 A1 EP2663323 A1 EP 2663323A1
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Prior art keywords
mir
nucleic acid
subject
complementary
levels
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EP2663323A4 (fr
EP2663323B1 (fr
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Anders M. Naar
Seyed Hani Najafi-Shoushtari
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General Hospital Corp
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General Hospital Corp
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • A—HUMAN NECESSITIES
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • This invention relates to methods for targeting microRNA-128 (miR-128) for regulating choiesterol/lipid metabolism and insulin sensitivity, inter alia.
  • LDL low-density lipoprotein
  • HDL high-density lipoprotein
  • Triglycerides as major components of very-low-density lipoprotein (VLDL), have been linked to atherosclerosis, and, by extension, the risk of heart disease and stroke. Elevated triglycerides (e.g., mildly elevated fasting levels, above 150 mg/dL (1.7 mmol/L), or high fasting levels above 200 mg/dL (2.26 mmol/L)) are common in subjects with metabolic syndrome/insulin resistance and those with poorly controlled diabetes, and contribute to the risk of atherosclerosis, heart disease, and. stroke in that population.
  • VLDL very-low-density lipoprotein
  • miR- 128-1 targets a large number of genes/proteins involved in cholesterol/lipid homeostasis and insulin signaling.
  • the data presented herein demonstrates that LDLR, ABCA1, SIRTl, and IRS1 are regulated by miR- 128-1.
  • antisense targeting of miR- 128-1 in human liver cells (HepG2) results in increased expression of these key cholestero ⁇ /lipid regulators and insulin signaling proteins.
  • Dys-reguladon of these proteins in humans is thought to contribute to aberrant cholesterol/lipid homeostasis and insulin resistance in Metabolic Syndrome and cardiovascular disease patients.
  • the methods described herein can be used to normalize cholesterol/lipid homeostasis and decrease insulin resistance.
  • the methods described herein include the use of inhibitory nucleic acids to target miR- 128-1 to improve cholesterol/lipid homeostasis and insulin resistance, e.g., in subjects with Metabolic Syndrome and/or
  • the invention provides methods for reducing levels of serum LDL, reducing levels of serum triglycerides, and/or increasing levels of serum HDL in a subject.
  • the methods include administering to the subject a therapeutically effective amount of an inhibitory nucleic acid that is complementary to all or part of any of SEQ ID NOs: 1-6, e.g., to all or part of SEQ ID NO:2.
  • the invention provides methods for treating or reducing the risk of developing diabetic neuropathy, non-alcoholic fatty liver disease, nonalcoholic steatohepatitis, atherosclerosis, and/or cardiovascular disease in a subject.
  • the methods include administering to the subject a therapeutically effective amount of an inhibitory nucleic acid that is complementary to all or part of any of SEQ ID NOs: 1 -6, e.g., to all or part of SEQ ID NO:2.
  • the invention provides methods for reducing obesity, treating or reducing predisposition to insulin resistance, and/or treating or reducing predisposition to type II diabetes, in a subject.
  • the methods include administering to the subject an inhibitory nucleic acid sequence that is complementary to all or part of any of SEQ ID NOs: 1-6, e.g., to all or part of SEQ ID NO:2, thereby decreasing obesity in the subject.
  • the invention provides methods for increasing uptake of lipids or cholesterol by a cell, e.g., a liver cell, or for increasing sensitivity of a cell, e.g., a liver cell, to insulin.
  • the methods include contacting the cell with an inhibitory nucleic acid sequence that is complementary to all or part of any of SEQ ID NOs: 1-6, e.g., to all or part of SEQ ID NO:2.
  • the inhibitory nucleic acid is complementary to at least nucleotides 2-7 (5'-CACAGU-3') of SEQ ID NO:3.
  • the inhibitory nucleic acid can be designed to target nucleotides 2-10 of the mature miR- 128-1 (SEQ ID NO:3), e.g., complementar to CACAGUGAA, e.g., have the sequence TTCACTGTG (SEQ ID NO:9, which is the same as nucleotides 12-20 of SEQ ID NO:7).
  • the inhibitory nucleic acid comprises all or part of AAAGAGACCGGTTC ACTGTGA (SEQ ID NO:7),
  • the inhibitor nucleic acid is an antisense oligonucleotide. In some embodiments, the antisense
  • oligonucleotide comprises a sequence that is complementary to SEQ ID NO: 3.
  • the inhibitory nucleic acid has one or more chemical modifications to the backbone or side chains as described herein. In some embodiments of the methods described herein, the inhibitory nucleic acid is an antagomir. In some embodiments of the methods described herein, the inhibitory nucleic acid has at least one locked nucleotide, and/or has a phosphorothioate backbone.
  • the inhibitory nucleic acid is an interfering RNA.
  • the interfering RNA is a small hairpin RNA (shRNA) or small interfering RNA (siRNA).
  • the inhibitory nucleic acid sequence inhibits post -transcriptional processing of SEQ ID NO: l or 5.
  • the subject has metabolic syndrome or Type 2 diabetes.
  • the methods include selecting a subject on the basis that they have metabolic syndrome or Type 2 diabetes.
  • the methods include detecting the presence of one or more alleles associated with increased levels of miR- 128 and/or predisposition to increased levels of serum lipids, and optionally selecting a subject on the basis of the presence of an allele associated with increased levels of miR-128.
  • the methods include determining a level of triglycerides in the subject, and selecting the subject if they have mildly elevated fasting levels (above 150 mg/dL (1.7 mmol/L) ⁇ or high fasting levels (above 200 mg/dL (2.26 mmol/L)).
  • the methods include selecting a subject who is in need of weight loss, e.g., a subject with a BMI of 25 or above.
  • a "complementary nucleic acid sequence” is a nucleic acid sequence capable of hybridizing with another nucleic acid sequence comprised of complementary nucleotide base pairs.
  • hybridize is meant pair to form a double- stranded molecule between complementary nucleotide bases (e.g., adenine (A) forms a base pair with thymine (T) (or uracil (U) in the case of RNA), and guanine (G) forms a base pair with cytosine (Q) under suitable conditions of stringency.
  • A adenine
  • T thymine
  • U uracil
  • G forms a base pair with cytosine (Q) under suitable conditions of stringency.
  • the inhibitory nucleic acid need not be complementary to the entire sequence, only enough of it to provide specific inhibition; for example in some embodiments the sequence is 100% complementary to at least nucleotides (nts) 2-7 or 2-8 at the 5' end of the microRNA itself (e.g. the 'seed sequence'), e.g., nts 2-7 or 2-8 of SEQ ID NOs:2 or 3. Further details are provided, below.
  • an "antisense oligonucleotide” refers to a nucleic acid sequence that is complementary to a D A or RNA sequence, such as that of a microRNA.
  • RNA is a molecule comprising at least one or more ribonucleotide residues.
  • a "ribonucleotide” is a nucleotide with a hydroxyl group at the 2' position of a beta- D- ribofuranose moiety.
  • the term RNA includes double-stranded RNA, single- stranded RNA, isolated RNA. such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA. as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Nucleotides of the RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotid.es.
  • miRNA is a single-stranded. RN A molecul e of about 21-23 nts in length. In general, miRNAs regulate gene expression. miRNAs are encoded by genes from whose DNA they are transcribed, but miRNAs are not translated into protein. Each primary miRNA transcript is processed into a short stem-loop structure (see, e.g., Fig. 3) before undergoing further processing into a functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to down-regulate gene expression.
  • mRNA messenger RNA
  • an interfering RNA refers to any double stranded or single stranded R.NA sequence, capable— either directly or indirectly (i.e., upon conversion) — of inhibiting or down regulating gene expression by mediating RNA interference.
  • Interfering RNA includes but is not limited to small interfering RNA ("siRNA”) and small hairpin RNA (“shRNA”).
  • siRNA small interfering RNA
  • shRNA small hairpin RNA
  • RNA interference' refers to the selective degradation of a sequence-compatible messenger RNA transcript.
  • an shRNA small hairpin RNA refers to an RNA molecule comprising an antisense region, a loop portion and a sense region, wherein the sense region has complementary nucleotides that base pair with the antisense region to form a duplex stem.
  • the small hairpin RNA is con verted into a small interfering RN A by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase HI family.
  • small interfering RNA or “siRNA” as used herein refers to any small RNA molecule capable of inhibiting or down regulating gene expression by mediating RNA interference in a sequence specific manner.
  • the small RNA can be, for example, about 18 to 21 nucleotides long.
  • an “antagomir” refers to a small synthetic RNA having complementarity to a specific microRNA target, with either mispairing at the cleavage site or one or more base modifications to inhibit cleavage.
  • post-transcriptional processing refers to mRNA processing that occurs after transcription and is mediated, for example, by the enzymes Dicer and/or Drosha.
  • an effective amount is meant the amount of a required agent or composition comprising the agent to ameliorate the symptoms of a disease relative to an untreated patient.
  • the effective amount of composition(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.”
  • cholesterol homeostasis refers to the regulation of cholesterol uptake, cholesterol biosynthesis, cholesterol conversion to bile acids and excretion of bile acids as such processes occur in a subject having healthful levels of LDL, HDL and cholesterol in the blood (e.g., such healthful levels are also referred to herein as a "reference standard"). Accordingly, a subject in need of cholesterol homeostasis is in need of improved, regulation resulting in a return to healthful levels of LDL, HDL and/or cholesterol in the blood.
  • a “subject” is a vertebrate, including any member of the class mammalia, including humans, domestic and farm animals, and zoo, sports or pet animals, such as mouse, rabbit, pig, sheep, goat, cattle and higher primates. In preferred embodiments the subject is a human.
  • a "vector” or “expression vector” is a nucleic acid-based delivery vehicle comprising regulatory sequences and a gene of interest, which can be used to transfer its contents into a cell.
  • FIG, 1 shows the sequence of human miR- 128- 1 precursor, also known as miR- 128A precursor (SEQ ID NO: l),
  • FIG. 2 shows the sequence of mature human miR-128 DNA (SEQ ID O:2) and RNA (SEQ ID NO:3). The mature sequences are the same for miR- 128-1 and miR-128-2.
  • FIG, 3 shows the predicted hairpin structure of miR-128-1 precursor RNA (SEQ ID NO:4).
  • FIG. 4 shows the sequence of human miR- 128-2 precursor, also known as miR-128B precursor (SEQ ID NO:5).
  • FIG. 5 shows the predicted hairpin structure of miR- 128-2 precursor RNA (SEQ ID NO:6).
  • FIG. 6 shows a schematic representation of the RAB3GAP 1 -R3HDM 1 -LC ' T genomic locus harboring miR- 128-1 on human chromosome 2.
  • miR-128- 1 is located in intron 18 of R3HDM1.
  • the depicted 1.23 Mb genomic region contains the S Ps associated with elevated LDL-C and TC in the analyses in Ma et al.
  • FIGs. 7A-B are bar graphs showing expression profiles of the miR-128-1 host gene R3HDM1 (7 A) and miR-128 (7B) in human tissues. Both genes are shown to be expressed fairly ubiquitously. To the best of the present inventors' knowledge, the function of R3HDM1 has not yet been determined. Additionally, the expression profile of miR-128 reflects the composite expression of both miR-128-1 (located in the R3HDM1 gene) and miR- 128-2, whose genomic location is within the ARPP-21 host gene. miR-128-1 and miR- 128-2 have identical mature sequences, which are measured here.
  • FIGs. 8A-B each show the immunoblotting results of three separate experiments demonstrating that introduction of anti sense oligonucleotides complementary to human miR-128-1 into human HepG2 liver ceils causes increased expression of LDLR (8 A), and introduction of human miR- 128-1 precursor oligonucleotides into human HepG2 liver cells causes decreased expression of LDLR (8B),
  • FIGs. 9A-B are each immunoblots showing that introduction of antisense oligonucleotides complementary to human miR-128-1 into human HepG2 liver cells causes increased expression of ABCA1 (9A), and introduction of human miR-128-1 precursor oligonucleotides into HepG2 ceils causes decreased expression of ABCA1 (9B).
  • miR-128-1 is shown to control the expression of ABCA 1.
  • FIGs. 10A-B are each immunoblots showing that introduction of antisense oligonucleotides complementary to human miR-128-1 into human HepG2 liver cells causes increased expression of SIRTl (10A), and introduction of human miR-128-1 precursor oligonucleotides into HepG2 ceils results in decreased expression of SIRTl (10B).
  • FIGs. 1 1A-C are immunoblots showing regulation of IR S I and CYP39A1 expression by miR-128-1.
  • Introduction of antisense oligonucleotides complementary to human miR-128-1 into HepG2 cells causes increased expression of IRS I (11 A), and introduction of human miR-128-1 precursor oligonucleotides into HepG2 cells causes decreased expression of IRSI (11 B) and decreased expression of CYP39A 1 (11C).
  • FIGs. 12A-B are bar graphs showing miR-128-1 target validation at LDLR- 3'UTR. Insertion of the LDLR-3'UTR sequence into a Luciferase reporter construct results in strongly decreased, luciferase expression in HEK293 cells, suggesting the presence of repressive regulatory motifs within the LDLR-3'UTR (12A).
  • FIGs. 13A-B are bar graphs showing that MiR-128-1 targets the ABCA1 (13A) and SIRTl (13B) 3' UTRs for post-transcriptionai regulation.
  • Introduction of human miR- 128-1 causes a repression of of the Luciferase-ABCAI and SIRTl 3'IJTRs, showing specific targeting by miR-128-1 through their 3'UTR.
  • FIGs. 14A-B are bar graphs showing that miR-128- i -mediated regulation of LDL receptor expression affects LDL uptake in human hepatoma cells.
  • Fluorescently labeled (Dil) LDL uptake is strongly reduced upon treatment of human HepG2 hepatoma cells with miR-128-1 precursors (14A), whereas a modest but significant increase in Dil-LDL uptake is observed after miR-128-1 antisense treatment (14B).
  • Ail experiments were repeated at least three times.
  • FIG. 15 is an immunoblot showing that miR-128-1 regulates ABCA1 and LDLR., which are involved in cholesteroi/lipid and energy homeostasis, in the human liver cell line Huh-7. miR- 128-1 antisense treatment of Huh-7 cells resulted in elevated expression of ABCA1 and LDLR. Beta-tubulin was used as negative control
  • FIG. 16 shows the results of Genome- wide analysis of miR-128-1 biological effect on lipoprotein metabolism genes in HepG2 human liver cells.
  • excess miR-128-1 decreases the level of its target gene LDLR
  • miR-128-1 also negatively affects the expression of major protein components of HDL lipoprotein particles such as ApoAl, ApoCl-3 and ApoE, indicating the disruptive role of miR- 128-1 in proper lipid metabolism and trafficking.
  • this indirect effect is extended to an induced expression of ApoB, a major component of VLDL and LDL lipoproteins ("bad cholesterol"). Unbiased hierarchical clustering analysis was applied to the DNA microarray data.
  • FIGs. 17A-B are line graphs showing that lend viral-mediated over-expression of miR- 128-1 increases the LDL/HDL ratio in mice.
  • FPLC analysis of cholesterol- containing lipoprotein (HDL and LDL) profiles in pooled serum samples from 5-10 mice each in two separate experiments indicate that ectopic miR-! 28-1 expression alters the distribution of serum HDL and LDL particles while total plasma cholesterol levels were not affected.
  • FIG. 18 shows the miR- .128-1 precursor sequence (bold.) including the miR- 128-1 guide-strand sequence (underlined.) flanked by 200 nt on each side, SEQ ID NO:8.
  • miR- 128-1 targets genes involved in cholesterol/lipid homeostasis and insulin signaling/energy homeostasis, suggest that the R3HDMl/miR-128- l genomic locus may harbor a "thrifty" gene whose elevated expression would allow increased fat storage/energy conservation in the face of starvation, thus providing a survival advantage in lean times.
  • increased activity of such a thrifty gene would also cause excess fat storage when food/energy resources are plenty, and could potentially contribute to Metabolic Syndrome, type 2 diabetes, and cardiovascular disease (CVD) in a subset of human populations harboring predisposing SNP alleles.
  • CVD cardiovascular disease
  • LDLR low-density lipoprotein receptor
  • ABCAl ATP- binding cassette A l transporter
  • HDL high-density lipoprotein
  • IRSl insulin signaling intermediate IRSl
  • CYF39A1 enzyme which converts cholesterol to bile for biliaxy excretion.
  • miR-128-1 antisense treatment e.g., in subjects suffering from Metabolic Syndrome (e.g., high LDL, low HDL, high triglycerides, obesity, nonalcoholic fatty liver disease, insulin resistance, a d/or hypertension), type 2 diabetes, and/or cardiovascular disease (CVD).
  • Metabolic Syndrome e.g., high LDL, low HDL, high triglycerides, obesity, nonalcoholic fatty liver disease, insulin resistance, a d/or hypertension
  • type 2 diabetes e.g., type 2 diabetes, and/or cardiovascular disease (CVD).
  • CVD cardiovascular disease
  • HDL due to elevated expression of ABCAl in liver and. peripheral tissues
  • miR-128- 1 may itself represent a central regulator of human metabolism rivaling transcription factors such as SREBPs, LXRs, PPARs, and CREB in governing diverse metabolic circuits.
  • the miR-128- 1 microRNA is located in intron 18 of the R3HDM1 gene on human chromosome 2.
  • Single nucleotide polymorphisms (SNPs) in a roughly 1 Mb genomic region in and surrounding the R3HDMl/miR-128-l locus have been associated with increased total cholesterol and LDL-cholesterol in genome-wide association studies (G WAS), including in the Framingham Heart Study and in a meta- analysis of 46 GWAS linking 95 genomic loci to various blood lipid parameters in >100,000 people.
  • MicroRNAs are a class of small (e.g., 1 8-24 nucleotides) non-coding RN As that exist in a variety of organisms, including mammals, and are conserved in evolution. miRNAs are processed from hairpin precursors of about 70 nucleotides which are derived trom primary transcripts through sequential cleavage by the RNAse III enzymes drosha and. dicer. Many microR As can be encoded, in intergenic regions, hosted, within introns of pre- mRNAs or within ncRNA genes. Many miRNAs also tend to be clustered, and.
  • MiRNAs have been found to have roles in a variety of biological processes including developmental timing, differentiation, apoptosis. cell proliferation, organ development, and metabolism.
  • the methods described herein include the inhibition miR-128 in a subject who has choiesterol/lipid abnormalities (e.g., elevated circulating LDL, low HDL, elevated triglycerides), and/or is insulin resistant, and/or has the metabolic syndrome, and/or is suffering from type 2 diabetes, and/or cardiovascular disease.
  • This can be achieved, for example, by administering an inhibitory nucleic acid, e.g., an antisense oligonucleotide that is complementary to miR-128. including but not limited to an aniisense oligonucleotide comprising all or part of
  • the oligo includes different modifications, e.g., in the sugar backbone, to make it more cell permeable and nuclease resistant on one hand, and physiologically non-toxic at lo concentrations on the other.
  • Other inhibitory nucleic acids for use in practicing the methods described herein and that are complementary to miR-128 can be those which inhibit post-transcriptional processing of miR-128, such as an interfering RNA, including but not limited to an shRNA or siR A, or an antagomir.
  • Inhibitory nucleic acids useful in the present methods and compositions include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, modified bases/locked nucleic acids (LNAs), antagomirs, peptide nucleic acids (PNAs), and other oligomeric compounds or oligonucleotide mimetics which hybridize to at least a portion of the target nucleic acid (i.e., miR-128, e.g., all or part of any of SEQ ID NOs: l -6) and modulate its function.
  • the target nucleic acid i.e., miR-128, e.g., all or part of any of SEQ ID NOs: l -6
  • the inhibitor ⁇ ' ' nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA- induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof.
  • RNAi interference RNA
  • siRNA short interfering RNA
  • miRNA micro, interfering RNA
  • shRNA small, temporal RNA
  • shRNA small RNA- induced gene activation
  • RNAa small activating RNAs
  • the inhibitory nucleic acids are 10 to 50, 13 to 50, or 13 to 30 nucleotides in length.
  • the oligonucleotides are 15 nucleotides in length.
  • the antisense or oligonucleotide compounds of the invention are 12 or 13 to 30 nucleotides in length.
  • One having ordinary skill in the art will appreciate that this embodies inhibitor ⁇ ' nucleic acids having antisense portions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or any range therewithin.
  • the inhibitory nucleic acids are designed to target a specific region of miR-128.
  • a specific functional region can be targeted, e.g., a region comprising a seed sequence or a region complementary to the target nucleic acid on which the miR-l 28 acts.
  • the mhibitory nucleic acid can be designed to target nucleotides 2-10 of the mature miR-l 28-1, e.g., complementary to CACAGUGAA, e.g., have the sequence TTCACTGTG (SEQ ID NO:9, which is the same as nucleotides 12-20 of SEQ ID O:7).
  • highly conserved regions can be targeted, e.g., regions identified by aligning sequences from disparate species such as primate (e.g., human) and rodent (e.g., mouse) and looking for regions with high degrees of identity .
  • Percent identity can be determined routinely using basic local alignment search tools (BLAS programs) (Altschul ei al,, J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), e.g., using the default parameters.
  • the inhibitory nucleic acids are chimeric
  • oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:D A or RNA:RNA hybrids.
  • Chimeric inhibitor ⁇ ' nucleic acids of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above.
  • Such compounds have also been referred to in the art as hybrids or gapmers.
  • Representative United States patents that teach the preparation of such hybrid structures comprise, but are not limited to, US patent nos. 5,013,830; 5, 149,797; 5, 220,007; 5,256,775; 5,366,878; 5,403,711 ; 5,491 ,133; 5,565,350; 5,623,065;
  • the inhibitory nucleic acid comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2 ! --Q-alkyl, 2 ! -0- alkyl-O-alkyl or 2'-fluoro-modified nucleotide.
  • RNA modifications include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3 ! end of the RNA. Such modifications are routinely incorporated into oligonucleotides and these
  • oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than; 2'-deoxyoligonucieotides against a given target.
  • modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain aikyl or cycloalkyi mtersugar linkages or short chain heteroaiomic or heterocyclic intersugar linkages. Most preferred are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH 2 -NH-0-CH 2 ,
  • CH, ⁇ N(CH 3 ) ⁇ Q ⁇ CH 2 (known as a methylene(methylimino) or MMI backbone], CH 2 --0--N (CH 3 )-CH 2 , CH 2 -N (CH 3 )-N (CH 3 )-CH 2 and O-N (CH 3 )- CH 2 -CH 2 backbones, wherein the native phosphodiester backbone is represented as O- P— O- CH,); amide backbones (see De Mesmaeker et ai. Ace. Chem. Res. 1995, 28:366- 374); morpholino backbone structures (see Summerton and Welier, U.S. Pat. No.
  • PNA peptide nucleic acid
  • Phosphorus- containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters,
  • ammoalkylphosphotriesters methyl and other alkyi phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkvlphosphotriesters, and boranophosphates having nonnal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted, polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3* or 2*-5* to 5'-2'; see US patent nos. 3,687,808; 4,469,863;
  • Morpbolino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41( 14), 4503-4510); Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et ah, Nat. Genet, 2000, 26, 216-220; Lacerra et a!., Proc. Nail. Acad. Sci., 2000, 97, 9591 -9596; and U.S. Fat. No. 5,034,506, issued Jul. 23, 1991.
  • Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc, 2000, 122, 8595-8602.
  • Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl
  • internucleoside linkages mixed eteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; suifamate backbones;
  • One or more substituted sugar moieties can also be included, e.g., one of the following at the 2 s position: OH, SH, SCH 3 , F, OCN, OCH 3 OCH 3 , OCH 3 0(CH 2 )n CH 3 , 0(CH 2 )n NH 2 or 0(CH 2 )n CH 3 where n is from 1 to about 10; Ci to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyi; CI; Br; CN; CF3 ; OCF3; 0-, S-, or N-alkyl; 0-, S-, or -alkenyl; SOCH3; S02 CH3; ON02; N02; N3; NH2; heterocycloalkyl; heterocycloalkar l; aminoalkylamino; polyalkylamino;
  • a preferred modification includes 2'- methoxyethoxy [2'-0-CH 2 CH 2 OCH 3 , also known as 2'-0-(2-methoxyethyl)] (Martin et af, Helv. Chim. Acta, 1995, 78, 486).
  • Oligonucleotides may also have sugar mimetics such as cyclobuty ls in place of the pentofuranosyl group.
  • Inhibitory nucleic acids can also include, additionally or alternatively, nucleobase (often referred to in the art simply as "base”) modifications or
  • nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5 -Me pyrimidines, particularly 5-methyleytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me- C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methyiamino)adenine, 2- (imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubsti
  • 2-aminoadenine 2- (methy
  • both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained, for hybridization with an appropriate nucleic acid, target compound.
  • an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone.
  • the nucieobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative United States patents that teach the preparation of PNA compounds comprise, but are not limited to. US patent nos.
  • Inhibitory nucleic acids can also include one or more nucieobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucieobases comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucieobases comprise other synthetic and natural nucieobases such as 5- methylcytosine (5-me-C), 5-hydroxymethyf cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2 -propyl and other alkyl deri vatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocyfosine, 5-halouracil and.
  • cytosine 5-propynyl uracil and.
  • cytosine 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracii), 4-thiouraciI, 8-halo. 8-amino, 8 -thiol, 8- thioalkyl, 8-hydroxyl and other 8 -substituted adenines and guanines, 5 -halo particularly 5- bromo, 5-irifluoromethyl and other 5-substiiuted.
  • uracils and cytosines 7-methylquamne and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine and 7-deazaadenine and 3- deazaguanine and. 3 -deaza adenine.
  • nucieobases comprise those disclosed in United States Patent No. 3,687,808, those disclosed in 'The Concise Encyclopedia of Polymer Science And Engineering', pages 858-859. Kroschwitz, J.I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al, Angewandle Chemie, International Edition', 1991 , 30, page 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications', pages 289- 302, Crooke, S.T. and Lebleu, B. ea., CRC Press, 1993. Certain of these nucieobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substiiuied.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6- 1.2 ⁇ 0>C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds, 'Antisense Research and 5 Applications', CRC Press, Boca Raton, 1993, pp. 276-278 ⁇ and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications. Modified nucleobases are described in US patent nos.
  • the inhibitory nucleic acids are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
  • moieties comprise but are not limited to, 5 lipid moieties such as a cholesterol moiety (Letsinger et al, Proc. Natl. Acad. Sci.
  • an aliphatic chain e.g., dodecandiol or undecyl residues (Kabanov et al, FEES Lett., 1990, 259, 327-330; Svinarchuk et al, Biochimie, 1993, 75, 49- 54), a phospholipid, e.g., di- exadecyi-rac-glycerol or triethylammonium I ,2-di-O- hexadecyl- rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al, Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a
  • conjugate groups of the invention include intercaiators, reporter molecules, poiyamines, poiyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
  • Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid.
  • Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No.
  • Conjugate moieties include, but are not limited, to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5- tritylthiol, a thioeholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadeeyl-rac- glycerol or triethylammonium 1,2-di-Q- hexadecyl-rae-giyeero-3-H-phosphonate, a poiyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octade
  • lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5
  • the inhibitory nucleic acids useful in the present methods are sufficiently complementary to all or part of miR-128, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect
  • “Complementary” refers to the capacity for pairing, through hydrogen bonding, between two sequences comprising naturally or non-naturally occurring bases or analogs thereof. For example, if a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a miR-128 sequence, then the bases are considered to be complementary to each other at that position. 100%
  • hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases.
  • adenine and thymine are complementary nucleobases which pair through the forma ti on of hydrogen bonds.
  • Complementary refers to the capacity for precise pairing between two nucleotides.
  • the inhibitory nucleic acids and the miR-128 are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other.
  • “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the inhibitory nucleic acid and the miR-128 target sequence. For example, if a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a miR-128 molecule, then the bases are considered to be complementary to each other at that position.
  • a complementary nucleic acid sequence need not be 100% complementary to that of its target nucleic acid to be specifically hybridisable.
  • a compleme tary nucleic acid sequence for purposes of the present methods is specifically hybridisable when binding of the sequence to the target miR- 128 molecule interferes with the normal function of the target miR-128 to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target miR-128 sequences under conditions in which specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency.
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and. more preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C.
  • Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed.
  • hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1 % SDS.
  • hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1 % SDS, 35% formamide, and 100 ⁇ °/ ⁇ denatured salmon sperm DNA (ssDN A).
  • hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 jig/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 rnM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C.
  • wash steps will occur at 25° C in 30 mM NaCL 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1 % SDS. In a more preferred embodiment, wash steps will occur at 68° C in 5 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
  • Hybridization techniques are well known to those skilled in the art and are described, for example, i Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley mterscience, New York, 2001 ⁇ ; Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and. Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
  • the inhibitory nucleic acids useful in the methods described herein have at least 80% sequence complementarity to a target region within the target nucleic acid, e.g., 90%, 95%, or 100% sequence complementarity to the target region within miR-128 (e.g., a target region comprising the seed sequence).
  • a target region within the target nucleic acid
  • an antisense compound in which 18 of 20 nucleobases of the antisense oligonucleotide are complementary, and. would therefore specifically hybridize, to a target region would represent 90 percent complementarity.
  • Percent complementarity of an inhibitory nucleic acid with a region of a target nucleic acid can be determined, routinely using basic local alignment search tools (BLAST programs) (Altschul et al., J.
  • Antisense and other compounds of the invention that hybridize to a miR-128 target sequence are identified through routine experimentation.
  • the inhibitory nucleic acids must retain specificity for their target, i.e., must not directly bind to, or directly significantly affect expression levels of, transcripts other than the intended target.
  • inhibitor ⁇ ' nucleic acids please see:
  • the inhibitory nucleic acids are antisense
  • Antisense oligonucleotides are typically designed to block expression of a DNA or RNA target by binding to the target and halting expression at the level of transcription, translation, or splicing.
  • Antisense oligonucleotides of the present invention are complementary nucleic acid sequences designed to hybridize under stringent conditions to a miR-128 target sequence. Thus, oligonucleotides are chosen that are sufficiently complementary to the target, i.e., that hybridize sufficiently well and with sufficient specificity, to give the desired effect.
  • the inhibitory nucleic acids used, in the methods described herein comprise one or more modified bonds or bases.
  • Modified bases include phosphorothioate, methylphosphonate, peptide nucleic acids, or locked nucleic acid (L A) molecules.
  • the modified nucleotides are locked nucleic acid molecules, including [alpha] -L-LNAs.
  • LNAs comprise ribonucleic acid analogues wherein the ribose ring is "locked" by a methylene bridge between the 2'- oxgygen and the 4'-carbon - i.e., oligonucleotides containing at least one LNA monomer, that is, one 2' ⁇ C?,4'- -methylene ⁇ ? ⁇ D-ribofaranosyl nucleotide.
  • LNA bases form standard Watson-Crick base pairs but the locked configuration increases the rate and stability of the basepairing reaction (Jepsen et al, Oligonucleotides, 14, 130-146 (2004)).
  • LN As also have increased affinity to base pair with RN A as compared to DNA.
  • LNAs especially useful as probes for fluorescence in situ hybridization (FISH) and comparative genomic hybridization, as knockdown tools for miRNAs, and as antisense oligonucleotides to target mRNAs or other RNAs.
  • the LNA molecules can include molecules comprising 10-30, e.g., 12-24, e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatched nucleotide(s), to a miR-128 target sequence.
  • the LNA molecules can be chemically synthesized using methods known in the art.
  • the LNA molecules cart be designed using any method known in the art; a number of algorithms are known, and are commercially available (e.g., on the internet, for example at exiqon.com). See, e.g., You et al, Nuc. Acids. Res. 34:e60 (2006); McTigue et al., Biochemistry 43:5388-405 (2004); and Levin et al, Nuc. Acids. Res. 34:el42 (2006).
  • “gene walk” methods can be used to optimize the inhibitory activity of the LNA (or any other inhibitory nucleic acid described herein); for example, a series of oligonucleotides of 10-30 nucleotides spanning the length of a target miR-128 sequence can be prepared, followed by testing for activity.
  • gaps e.g., of 5-10 nucleotides or more, can be left between the LNAs to reduce the number of oligonucleotides synthesized and tested.
  • GC content is preferably between about 30-60%.
  • LNA sequences will bind very tightly to other LNA sequences, so it is preferable to avoid significant complementarity within an LN A. Contiguous runs of three or more Gs or Cs, or more than four LNA residues, should be avoided where possible (for example, it may not be possible with very short (e.g., about 9-10 nt) oligonucleotides).
  • the LNAs are xyfo-LNAs.
  • the inhibitory nucleic acid is or comprises TTCACTGTG (SEQ ID NO: 9), wherein all of the nucleic acids are locked and the backbone is a phosphorothioate backbone,
  • the antisense is an antagomir.
  • Antagomirs are chemically modified antisense oligonucleotides that target a miR-128 target sequence.
  • an antagomir for use in the methods described, herein can include a nucleotide sequence sufficiently complementary to hybridize to a miR- 128 target sequence of about 12 to 25 nucleotides, preferably about 15 to 23 nucleotides.
  • antagomirs include a cholesterol moiety, e.g., at the 3'-end,
  • antagomirs have various modifications for RNase protection and pharmacologic properties such as enhanced tissue and cellular uptake.
  • an antagomir can have one or more of complete or partial 2'-G-methylation of sugar and/or a phosphorothioate backbone.
  • the antagomir can include six phosphorothioate backbone modifications; two phosphorothioates are located at the 5'-end and four at the 3'-end. See, e.g., KriUzfefdt et al, Nature 438, 685-689 (2005); Czech, N Engl J Med 2006; 354: 1 194- 1 195 (2006); Robertson et al., Silence. 1 : 10 (2010); Marquez and
  • Antagomirs useful in the present methods can also be modified with respect to their length or otherwise the number of nucleotides making up the antagomir.
  • the antagomirs must retain specificity for their target, i.e., must not directly bind to, or directly
  • transcripts other than the intended target significantly affect expression levels of, transcripts other than the intended target.
  • the inhibitory nucleic acid is locked and includes a cholesterol moiety (e.g., a locked antagomir).
  • a cholesterol moiety e.g., a locked antagomir.
  • the nucleic acid sequence that is complementary to miR-128 can be an interfering RNA. including but not limited to a small interfering RNA ("siRNA”) or a small hairpin RNA (“shRNA").
  • interfering RN can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self- complementary (i.e., each strand comprises nucleotide sequence that is
  • the antisense strand and sense strand form a duplex or double stranded structure
  • the antisense strand comprises nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (i.e., an undesired gene) and.
  • the sense strand comprises nucleotide sequence corresponding to the target nucleic acid. sequence or a portion thereof.
  • interfering RNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions are Jinked by means of nucleic acid based or non-nucleic acid-based, Imker(s).
  • the interfering RNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the interfering can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and.
  • the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siR A molecule capable of mediating RNA interference.
  • the interfering RNA coding region encodes a self- complementary RNA molecule having a sense region, an antisense region and a loop region.
  • a self- complementary RNA molecule having a sense region, an antisense region and a loop region.
  • Such an RNA molecule when expressed desirably forms a "hairpin" structure, and is referred to herein as an "shR A.”
  • the loop region is generally between about 2 and about 10 nucleotides in length. In some embodiments, the loop region is from about 6 to about 9 nucleotides in length.
  • the sense region and the antisense region are between about 15 and about 20 nucleotides in length.
  • the small hairpin RNA is converted into a siRNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family.
  • Dicer which is a member of the RNase III family.
  • the siRNA is then capable of inhibiting the expression of a gene with which it shares homology. For details, see Brummelkamp et al., Science 296:550-553, (2002); Lee et al, Nature Biotechnol., 20, 500-505, (2002); Miyagishi and Taira, Nature Biotechnol 20:497-500, (2002); Paddison et ai. Genes & Dev.
  • siRNAs The target RNA cleavage reaction guided by siRNAs is highly sequence specific.
  • siRNA containing a nucleotide sequences identical to a portion of the target nucleic acid are preferred for inhibition.
  • 100% sequence identity between the siRNA and the target gene is not required to practice the present invention.
  • the invention has the advantage of being able to tolerate sequence variations that might be expected, due to genetic mutation, strain polymorphism, or evolutionary divergence.
  • siRNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition.
  • siRNA sequences with nucleotide analog substitutions or insertions can be effective for inhibition.
  • the siRNAs must retain specificity for their target, i.e., must not directly bind to, or directly
  • Trans-cleaving enzymatic nucleic acid molecules can also be used; they have shown promise as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med, Chem. 38, 2023-2037).
  • Enzymatic nucleic acid molecules can be designed to cleave mill- 128 within the background of cellular RNA. Such a cleavage event renders the miR- 128 non- functional.
  • enzymatic nucleic acids with RNA cleaving activity act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which, is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA.
  • the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base pairing, and once bound to the correct site, acts enzymatieally to cut the target RNA. Strategic cleavage of such a target RN A will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
  • RNA, cDNA, genomic DNA, vectors, viruses or hybrids thereof can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed'' generated recombinantly.
  • Recombinant nucleic acid sequences can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including e.g. in vitro, bacterial, fungal, mammalian, yeast, insect or plant cell expression systems.
  • Nucleic acid sequences of the invention can be inserted into delivery vectors and expressed from transcription units within the vectors.
  • the recombinant vectors can be DNA plasmids or viral vectors.
  • Generation of the vector construct can be accomplished using any suitable genetic engineering techniques well known in the art, including, without limitation, the standard techniques of PGR, oligonucleotide synthesis, restriction endonucfease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al.
  • RNA Viruses A Practical Approach
  • suitable vectors are available for transferring nucleic acids of the invention into cells.
  • Viral vectors comprise a nucleotide sequence having sequences for the production of recombinant virus in a packaging cell.
  • Viral vectors expressing nucleic acids of the invention can be constructed based on viral backbones including, but not limited to, a retrovirus, lentivirus, adenovirus, adeno-associated virus, pox virus or alphavirus.
  • the recombinant vectors capable of expressing the nucleic acids of the invention can be delivered as described herein, and persist in target cells (e.g., stable transformants).
  • Nucleic acid sequences used to practice this invention can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661 ; Belousov (1997) Nucleic Acids Res. 25:3440 » 3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896: Narang (1979) Meth. Enzymol. 68:90: Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981 ) Tetra. Lett. 22: 1859; U.S. Patent No. 4,458,066.
  • nucleic acid sequences of the invention can be stabilized against nueleolytic degradation such as by the incorporation of a modification, e.g.. a nucleotide modification.
  • nucleic acid sequences of the invention includes a phosphorothioate at least the first, second, or third, mteraucfeotide linkage at the 5' or 3' end of the nucleotide sequence.
  • the nucleic acid sequence can include a 2 !
  • nucleotide e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-G-methyl, - O-methoxyethyl (2'-0-MQE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0- dimethylaniinoethyloxyethyl (2 -O-DMAEOE), or 2'-0— -methylacetamido (2 -0- NMA).
  • the nucleic acid sequence can include at least one 2 -0- methyl -modified nucleotide, and in some embodiments, all of the nucleotides include a 2'-0-methyl modification.
  • the nucleic acids are "locked," i.e., comprise nucleic acid analogues in which the ribose ring is "locked” by a methylene bridge connecting the 2'-0 atom and. the 4'-C atom (see, e.g., Kaupinnen et al., Drug Disc. Today 2(3):287-290 (2005); Koshkin et al., J. Am. Chem. Soc, 120(50): 13252-13253 ( 1998)).
  • For additional modifications see US 20100004320, US 20090298916, and US 20090143326.
  • nucleic acids used to practice this invention such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 3d ed. (2001); Current Protocols in Molecular Biology, Ausubel et al., eds. (John Wiley & Sons, Inc., New York 2010); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); Laboratory Techniques In Biochemistry And Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed.
  • labeling probes e.g., random-primer labeling using Klenow polymerase, nick translation, amplification
  • sequencing hybridization and the like
  • the methods described herein can include the administration of
  • compositions and. formulations comprising inhibitor ⁇ ' ' nucleic acid sequences designed to target miR-128.
  • compositions are formulated with a
  • compositions and formulations can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermaily.
  • the pharmaceutical compositions and formulations can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermaily.
  • compositions can be formulated in any way and can be administered in a variety of unit dosage fonns depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see, e.g., Rem ingto ; The Science an d Prac ti ce of Pharmacy, 21 st ed., 2005.
  • the inhibitory nucleic acids can be administered alone or as a component of a pharmaceutical formulation (composition).
  • composition may be formulated for administration, in any convenient way for use in human or veterinary medicine.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • Formulations of the compositions of the invention include those suitable for intradermal, inhalation, oral/ nasal, topical, parenteral, rectal, and/or intravaginal administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient (e.g., nucleic acid sequences of this invention) which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, e.g., intradermal or inhalation.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect, e.g., an antigen specific T cell or humoral response.
  • compositions of this invention can be prepared, according to any method known to the art for the manufacture of pharmaceuticals.
  • Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents.
  • a formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture.
  • Formulations may comprise one or more diluents, emulsifters, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient.
  • Pharmaceutical preparations for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores.
  • Suitable solid, excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitoi, or sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-celiulose, or sodium carboxy-methylcellulose; and gums including arable and tragacanth; and proteins, e.g., gelatin and collagen.
  • Disintegrating or soiubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Push-fit capsules can contain active agents mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active agents can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
  • Aqueous suspensions can contain an active agent (e.g., nucleic acid sequences of the invention) in admixture with excipients suitable for the manufacture of aqueous suspensions, e.g., for aqueous intradermal injections.
  • excipients include a suspending agent, such as sodium carboxymefhylcellulose, methylcellulose, hydroxypropylmethyl cellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g..
  • lecithin a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyefhylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., he tadecaethylene oxyeeta.no!), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitoi (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitoi anhydride (e.g., polyoxyethylene sorbitan mono-oieate).
  • a condensation product of an alkylene oxide with a fatty acid e.g., polyoxyefhylene stearate
  • a condensation product of ethylene oxide with a long chain aliphatic alcohol e.g., he tadecaethylene
  • the aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin.
  • preservatives such as ethyl or n-propyl p-hydroxybenzoate
  • coloring agents such as a coloring agent
  • flavoring agents such as aqueous suspension
  • sweetening agents such as sucrose, aspartame or saccharin.
  • Formulations can be adjusted for osmolality.
  • oil-based pharmaceuticals are used for administration of nucleic acid sequences of the invention.
  • Oil-based suspensions can be formulated by suspending an active agent in a vegetable oil. such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. See e.g., U.S. Patent No, 5,716,928 describing using essential oils or essentia oil components for increasing bioavailability and reducing inter- and infra-individual variability of orally administered hydrophobic pharmaceutical compounds (see also U.S. Patent No. 5,858,401 ).
  • the oil suspensions can contain a thickening agent, such as beeswax, hard, paraffin or cetyl alcohol.
  • Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid.
  • an injectable oil vehicle see Minto (1.997) J. Pharmacol. Exp. Ther. 281 :93-102.
  • compositions can also be in the form of oil-in-water emulsions.
  • the oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these.
  • Suitable emulsifying agents include naturally- occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitoi anhydrides, such as sorbitan mono-oleate, and. condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate.
  • the emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs.
  • Such formulations can also contain a demulcent, a preservative, or a coloring agent.
  • these injectable oil -in- water emulsions of the invention comprise a paraffin oil, a sorbitan monooleate, an ethoxylated sorbitan monooieate and/or an ethoxylated sorbitan trioleate.
  • the pharmaceutical compounds can also be administered by in intranasal, intraocular and intravagmal routes including suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see e.g., Rohatagi (1995) J. Clin. Pharmacol. 35; 1187-1 193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75: 107- 1 1 1 ⁇ .
  • Suppositories formulations can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug.
  • suitable non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug.
  • Such materials are cocoa butter and polyethylene glycols.
  • the pharmaceutical compounds can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
  • the pharmaceutical compounds can also be delivered, as microspheres for slow release in the body.
  • microspheres can be administered via intradermal injection of drug which slowly release subcutaneously: see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oral administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.
  • the pharmaceutical compounds can be parenterally administered, such as by intravenous (IV) administration or administration into a body cavity or lumen of an organ.
  • IV intravenous
  • These formulations can comprise a solution of active agent dissolved in a pharmaceutically acceptable carrier.
  • Acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride.
  • sterile fixed oils can be employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter.
  • These formulations may be sterilized by conventional, well known sterilization techniques.
  • the formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of active agent in these formulations can var widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs.
  • the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation can also be a suspension in a nontoxic parenterally-aceeptable diluent or solvent, such as a solution of 1,3- butanediol.
  • the administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time).
  • the pharmaceutical compounds and formulations can be lyophilized.
  • Stable lyophilized formulations comprising an inhibitory nucleic acid can be made by lyophiiizing a solution comprising a pharmaceutical of the invention and a bulking agent, e.g., mannitoi, trehalose, raffmose, and sucrose or mixtures thereof.
  • a process for preparing a stable lyophilized formulation can include lyophiiizing a solution about 2.5 mg mL protein, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. See, e.g., U.S. 20040028670.
  • compositions and formulations can be delivered by the use of liposomes.
  • liposomes particularly where the liposome surface carries ligands specific for target ceils, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Patent Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13:293-306; Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46: 1576-1587.
  • liposome means a vesicle composed of amphophilic lipids arranged in a bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed, to entrap DNA rather than complex with it. Both cationic and. noncationic liposomes have been used to deliver DNA to cells.
  • Liposomes can also include "sterically stabilized" liposomes, i.e., liposomes comprising one or more specialized lipids. When incorporated, into liposomes, these specialized, lipids result in liposomes with enhanced, circulation lifetimes relative to liposomes lacking such specialized lipids.
  • sterically stabilized liposomes are those in which part of the vesicle-forming lipid, portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
  • PEG polyethylene glycol
  • compositions of the invention can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a subject who is need of reduced triglyceride levels, or who is at risk of or has a disorder described herein, in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the disorder or its complications; this can be called a therapeutically effective amount.
  • pharmaceutical compositions of the invention are administered in an amount sufficient to decrease serum levels of triglycerides in the subject.
  • the amount of pharmaceutical composition adequate to accomplish this is a therapeutically effective dose.
  • the dosage schedule and. amounts effective for this use i.e., the dosing regimen, will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.
  • the dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo- Aragon.es (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pliarmazie 51 :337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm, Sci. 84: 1144- 1146: Rohatagi (1995) Fhamazie 50:610-613; Brophy (1983) Eur, ,1. Clin. Pharmacol.
  • formulations can be given depending on for example: the dosage and frequency as required and tolerated by the patient, the degree and amount of therapeutic effect generated after each administration (e.g., effect on rumor size or growth), and the like.
  • the formulations should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate conditions, diseases or symptoms.
  • administration are in a daily amount of between about 1 to 100 or more mg per kilogram of body weight per day.
  • Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ.
  • Substantially higher dosages can be used in topical or oral administration or administering by powders, spray or inhalation.
  • Actual methods for preparing parenterally or non-parentera31y administrable formulations will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington: The Science and. Practice of Pharmacy, 21st ed. , 2005.
  • LNAs locked nucleic acids
  • the methods described herein can include coadministration with other drugs or pharmaceuticals, e.g., compositions for providing cholesterol homeostasis.
  • the inhibitory nucleic acids can be coadministered with drugs for treating or reducing risk of a disorder described herein.
  • the methods can also include identifying subjects for treatment using a method described by detecting one or both of (i) the presence of a genetic variation that predisposes a subject to elevated miR-128 levels, or (ii) the presence of elevated miR-128 levels, e.g., in a sample comprising liver and/or white adipose cells or tissue, e.g., from a biopsy.
  • detecting the presence of a genetic variation that predisposes a subject to elevated miR-128 levels includes detecting the identity of an allele of one, two or more of rs6730! 57, rs! 2465802, rs4954280, rs2322660, rs309180, rs632632, rs7570971 , rs4988235, or rs6719488, wherein the presence of an allele that is associated with elevated levels of miR-128, or with increased risk of elevated lipid levels (e.g., as described in Ma et al., BMC Medical Genetics 1 1 :55 (2010); Teslovich et al, Nature 466:707-713 (2010); and Silander et ah, PLoS One 3 3615 (2008)), indicates that the subject is predisposed to have elevated levels of miR-128, and thus would benefit from the treatment methods described herein.
  • the methods include selecting a subject for treatment with a method
  • Abnormal cholesterol and triglyceride levels are associated with risk of disease, including cardiovascular disease; in some embodiments, the subjects treated using the methods described herein have, or are at risk of developing, abnormal cholesterol and/or triglyceride levels. In some embodiments, the subjects have elevated LDL-cholesterol, elevated total cholesterol, and/or elevated triglyceride levels.
  • High Density Lipoprotein (HDL), Low Density Lipoprotein (LDL) and Ver Low Density Lipoprotein (VLDL) are the three major kinds of cholesterol that are monitored.. Total cholesterol and cholesterol/HDL ratio can also be monitored.
  • the following tables provide information regarding levels of cholesterol that are considered to be optimal (i.e., associated with a low or normal risk of cardiovascular disease) or abnormal (i.e., associated with a higher risk of cardiovascular disease).
  • the numbers are in milligrams per deciliter (mg/dL).
  • Elevated levels of LDL are associated with increased risk of cardiovascular disease.
  • optimal LDL levels In the presence of cardiovascular disease, some experts consider optimal LDL levels to be less than 70. For people with diabetes or other multiple risk factors for heart disease, optimal le vels of LDL are less than 100. Increased, levels of HDL cholesterol are associated with decreased risk of cardiovascular disease.
  • Total blood cholesterol is a measure of LDL cholesterol, HDL cholesterol, and other lipid components combined. Total cholesterol levels below 200 are desirable.
  • Triglyceride (triacylglycerol, TAG or triacylglyceride) is an ester derived, from glycerol and three fatty acids, and is the main constituent of vegetable oil and animal fats (Nelson, D. L.; Cox, M. M. Lehninger, Principles of Biochemistry . 3rd Ed. Worth Publishing: New York, 2000).
  • Fasting triglyceride levels can be determined using any means known in the art, e.g., enzymaticaily using a glycerol kinase reaction-based coiorimetric assay. Cholesterol levels can also be determined using any means known in the art, e.g., using immunoassay, electrophoresis, NMR, and/or precipitation-based methods.
  • the subjects treated, by the methods described herein have diabetes, i.e., are diabetic.
  • a person who is diabetic has one or more of a Fasting Plasma Glucose Test result of 126 mg/dL or more; a 2-Hour Plasma Glucose Result in an Oral Glucose Tolerance Test of 200 mg/dL or more; and blood glucose level of 200 mg/dL or above.
  • the subjects treated by the methods described herein are being treated for diabetes, e.g., have been prescribed or are taking insulin, m eg! ⁇ tin ides, biguanides, thiazolidinediones, or alpha-glucosidase inhibitors.
  • the subjects are pre-diabetic, e.g., they have impaired glucose tolerance or impaired fasting glucose, e.g., as determined by standard clinical methods such as the intravenous glucose tolerance test (IVGTT) or oral glucose tolerance test (OGTT), e.g., a value of 7.8-11.0 mmol L two hours after a 75 g glucose drink for impaired glucose tolerance, or a fasting glucose level (e.g., before breakfast) of 6.1-6.9 mmol/L.
  • IVGTT intravenous glucose tolerance test
  • OGTT oral glucose tolerance test
  • the methods described herein include selecting subjects who have diabetes or pre-diabetes.
  • the following table is used to identify and/or select subjects who are diabetic or have pre-diabetes, i.e., impaired glucose tolerance and/ or impaired fasting glucose. Fasting Blood Glucose
  • the methods described herein can include determining a subject's height, determining a subject's weight, and calculating BMI from the values determined thereby.
  • the methods described herein can include reviewing a subject's medical history to determine their BMI.
  • the methods described herein include selecting subjects who have a BMI of 30 or above (i.e., obese subjects ⁇ . Metabolic Syndrome
  • the methods include determining whether a subject has the metabolic syndrome, and selecting the subject if they do have the metabolic syndrome, then administering an inhibitor ⁇ '' nucleic acid as described herein.
  • Determining whether a subject has the metabolic syndrome can include reviewing their medical history, or ordering or performing such tests as are necessary to establish a diagnosis.
  • the metabolic syndrome refers to a clustering of obesity, dyslipidemia, hypertension, and insulin resistance. All components of the metabolic syndrome are traditional risk factors for vascular disease.
  • the metabolic syndrome is defined by the presence of at least 3 of the following: abdominal obesity (excessive fat tissue in and around the abdomen, as measured by waist circumference:
  • fasting blood triglycerides e.g., greater than or equal to 150 mg/dL
  • low blood HDL e.g., less than 40 mg/dL for men, and less than 50 mg/dL for women
  • elevated LDL e.g., above 130 mg/dL
  • high blood pressure e.g., greater than or equal to 130/85 nimHg
  • elevated fasting glucose e.g., greater than or equal to 110 mg/dL.
  • levels of these criteria may be higher or lo was, depending on the subject; for example, in subjects of Asian ancestry; see, e.g., Meigs, Curr. Op.
  • a determination of the presence of metabolic syndrome can be made, e.g., by reviewing the subject's medical history, or by reviewing test results.
  • NHANES 111 Based on data from the Third National Health a d Nutrition Examination Survey (NHANES 111) approximately 24% of the adults in the United States qualify as having the metabolic syndrome (Ford et al, JAMA. 287(3):356-359 (2002)). Insulin resistance is now felt to be central in the pathogenesis of these related disorders.
  • Nonalcoholic fatty liver disease and its most severe form, nonalcoholic steatohepatitis (NASH), are associated with high fat diet, high triglyceride levels, obesity, the metabolic syndrome and. type 2 diabetes, and pose an increased risk of cardiovascular disease.
  • NAFLD is an accumulation of fat in the liver that is not a result of excessive consumption of alcohol. 15% to 25% of cases of NAFLD progress and are associated with inflammation and liver damage; this condition is referred to as NASH.
  • NASH is associated with an increased risk of developing liver cirrhosis and subsequence complications, including hepatocellular carcinoma.
  • a diagnosis of N AFLD or NASH can be made by methods known in the art, e.g., by histological examination of liver biopsy samples.
  • Example 1 A Functional Role of miR-128 in Lipid Homeostasis and Insulin Signaling
  • miR-128 plays a role in lipid homeostasis and insulin signalmg.
  • expression of miR-128- 1 was evaluated in 20 selected human tissues using the Taqman miRNA Assay Kit (Applied Biosystems). Total RNA samples were purchased from Ambion, now an Applied Biosystems company. Briefly, 10 ng total RNA was reverse-transcribed using a miR-128- specific primer (comprising sequence complementary to part of SEQ NO:3), followed by quantitative real-time PGR with Taqman probes. Thereby, the U6 sp!iceosomaf RN was used, as an internal control.
  • miR-128-1 was co-expressed with the host gene R3HDM1 in a number of human tissues (Figs. 7A-B), Both genes were shown to be expressed fairly ubiquitously. The function of R3HDM1 has not yet been determined.
  • the expression profile of miR- 128 shown in Fig. 8B reflects the combined expression of miR-128-1 (located in the R3HDM1 gene) and miR-128-2, whose genomic location is within the ARPP-21 host gene. miR-128-1 and miR-128-2 have identical mature sequences, which are measured here.
  • miR-128 plays a role in regulating lipid homeostasis and insulin signalmg
  • experiments were performed to see what effect increased or decreased expression of miR-128 would have on genes associa ted, with lipid homeostasis, including LDLR, ABCAl, SIRT1, and CYP39A1, as well as the insulin-signaling component 1RS1.
  • Mutations in LDLR cause the autosomal dominant disorder familial hypercholesterolemia (Brown and Goldstein, Scientific American, 1984, 252:52-60; Leigh SE et al.
  • the ABCAl protein functions as a cholesterol efflux pump in the cellular lipid removal pathway, thereby promoting HDL biosynthesis. Mutations in the ABCAl gene have been associated, with Tangier's disease, familial high-density lipoprotein deficiency, and elevated cardiovascular disease risk (Brooks- Wilson et al, (1999) Nature Genetics, 22:336-45; Hong et al, (2003) Atherosclerosis 164: 245-250).
  • the CYP39A 1 enzyme contributes to the cholesterol catabolic pathway in the liver, which converts cholesterol to bile acids, the primary mechanism for the removal of excess cholesterol from the body. Mutations in this CYP subfamily of enzymes are associated with defects in bile acid biosynthesis which can eventually lead to severe metabolic disorders (Wang et al., (2009) J Lipid Res.
  • SIRT1 regulates the function of many key transcription factors in human metabolism. For example, it inhibits SREBP activity, a master regulator of cholesterol and fat metabolism (Walker et al, (2010) Genes Dev. 24: 1403-1417).
  • IRSl plays a key role in transmitting signals from the insulin receptor to intracellular metabolic pathways. Mutations in the IRSl gene are associated with type II diabetes and susceptibility to insulin resistance (Berger et al. (2002), Diabet Med. 19:804-809; Eftychi Get al. (2004) Diabetes. 53:870-873).
  • rniR- 128 The effects of increasing expression of rniR- 128 were evaluated by the introduction of excess rniR- 128-1 precursor oligonucleotides into human HepG2 liver cells.
  • the precursor oligonucleotides were double stranded, and. mimicked, the precursor sequence of miR-128.
  • the active strand comprises the sequence ID NO.3.
  • Antisense treated cells were harvested 24 hours after transfection, whereas cells treated with Precursor oligonucleotides were harvested after 48 hours of transfection in whole cell extract buffer, Immunoblotting was carried out using protein specific antibodies according to manufacturer's protocol.
  • Antibodies for LDLR, ABCAl and CYP39A1 were from Abeam.
  • SIRT1 antibody was from Cyelex, and the antibody for IRSl from Cell Signaling,
  • Figs 8-1 increasing expression of miR-128 results in decreased expression of key cholesterol/lipid regulators such as LDLR, ABCAl , SIRT1, and CYP39A 1 , as well as the insulin-signaling component IRSl .
  • key cholesterol/lipid regulators such as LDLR, ABCAl , SIRT1, and CYP39A 1 .
  • rniR- .128-1 antisense oligonucleotides were evaluated by the introduction of excess rniR- .128-1 antisense oligonucleotides into human HepG2 Hver cells.
  • the antisense oiigos were purchased from Ambion (Applied Biosystems). Transfection studies were all carried out by electroporation using cell-specific transfection reagents from Lonza Company. Each transfection procedure contained 2x10° HepG2 cells and 0.1 nmol of antisense oligo. Upon transfection, cells were plated on polylysine-coated plates and harvested for analysis 24 hours post incubation at 37°C under 5% C02. Cells were all grown in MEM medium with 10% FBS.
  • the antisense sequence used in this study was AAAGAGACCGGTTCACTGTGA (SEQ ID NO: 7). According to the manufacturer the oligo contains a phosphorothioate backbone and 2'-Methoxy moieties.
  • a reporter construct was developed that included the LDLR-3 ' UTR sequence Jinked to a luciferase reporter.
  • the construct contained the entire LDLR 3 'UTR sequence (2498 bp) downstream of the Luciferase gene (RenSP).
  • Transfections were carried out using the LIPOFECTAMINE transfection reagent.
  • HEK293 cells were transfected with 10 ng of plasmid construct and 30 nM of Precursor miR-128 oiigos, and the Luciferase actvity was measured 24 hours post-transfection.
  • a beta-Galactosidase expression vector was cotransfected (100 ng) and its activity used as a normalizer.
  • LDLR 3 'UTR When expressed in HEK 293 cells, the LDLR 3 'UTR mediated strong repression of the fused Luciferase reporter (Fig. 12A), and this repression was further increased upon introduction of additional miR-128-1 precursor oligonucleotides (Fig. 12B).
  • lipid uptake human liver cells (HepG2) were treated with the antisense and precursor oiigos as described above.
  • the LDL uptake experiment was initiated by adding Di3-LDL (Biomedical Technologies) in a final concentration of 10 ⁇ &' ⁇ . The LDL uptake was carried out for 2 hours. Upon incubation, ceils were washed three times with cold PBS. Cells were finally lysed in SS buffer containing 0.1 M NaOH and 0.1% SDS according to standard protocols (Stephan ZF & Yurachek EC, (1993) J Lipid Res 34: 325-330).
  • miR- 128-1 is an important regulator of hepatic LDLR expression and LDL uptake.
  • miR- 128-1 is the first significant regulator of LDL expression since the discovery of the SREBP genes 17 years ago by Brown and Goldstein at LIT Scontaminated Medical Center in Dallas (Yokoyama et al., ( 1993) Ceil, 75: 187-195; Goldstein and Brown, (2009) Arterioscler Thromb Vase Biol. 29:431 -438).
  • ABCA 1 cholesterol transporter is another potential target of miR-128-1 (Table 1).
  • ABCA 1 is critical for the production of HDL by the liver, and also acts as a cholesterol efflux pump that extrudes cholesterol and phospholipids to HDL, from peripheral tissues and cells, including arterial macrophages, to promote reverse cholesterol transport (RCT) (Cuchel and Rader, Circulation 1 13 (21 ), 2548-2555 (2006)).
  • RCT reverse cholesterol transport
  • variations in the ABCA1 gene have been linked to impaired HDL synthesis and RCT, and may be associated with elevated risk for atherosclerosis (Rader et al, J Lipid Res 50 Suppl, S I 89 ⁇ 1 4 (2009)).
  • SIRTl regulates the function of many key transcription factors in human metabolism. For example, it inhibits SREBP activity, a master regulator of cholesterol and fat metabolism (Walker et al., (2010) Genes Dev. 24: 1403-1417).
  • reporter constructs were developed that included either the ABCAl-3' UTR and SIRTl -3' UTR sequence linked to a luciferase reporter.
  • the constructs contained the SIRTl or ABCA1 3 'UTR sequences downstream of the Luciferase gene (RenSP).
  • Pre-plated HEK293 cells were transfected with 40 nM miR-128-1 precursor or precursor control (Ambion) in the presence of 2 ng of ABCA1 or SIRTl luciferase plasmid constructs, respectively, using lipofectamine 2000 (Invitrogen/Life Technologies).
  • Luciferase-ABCA l (Fig. 13A) and SIRTl (Fig. 13B) 3'UTRs, showing specific targeting by miR-128-1 through their 3'UTR.
  • Huh-7 cells were treated with 50 nM of Anti-miR-128 - 1 (Ambion) and plated at high density in DMEM media containing 5% FBS. After 48 hrs ceils were harvested and protein amount was quantified by western blotting analysis. As shown in Fig. 15, coordinated up regulation of ABCA1 and LDLR in Huh-7 human liver cells was seen in response to miR-128-1 antisense inhibition. Thus, miR-128-1 indeed controls the levels of ABCA1 in human liver cells (Fig. 15).
  • MicroRNAs typically regulate many targets, frequently by controlling mRNA stability 48 .
  • TargetScan provides a mathematical predictor of microR A targets.
  • DNA microarray analysis was employed to obtain a comprehensive list of potential miR-128-1 targets. These studies identify novel pathways regulated by miR- 128-1 that may affect therapeutic targeting efforts. Samples were analyzed on Affymetrix human genome U133 Plus 2.0 arrays by Affymetrix readers.
  • miR-128-1 manipulation will affect lipid/cholesterol storage and energy usage in mice, and that the effects of miR-128-1 overexpression will be exacerbated by a high-fat diet while antisense treatment will provide protection from deleterious effects normally associated, with this diet.
  • SBI System Biosciences Inc.
  • the modified pMIRNAl consists of the native human miR-128-1 stem loop structure and 200-400 base pairs of upstream and downstream flanking genomic sequence (see Fig 18, SEQ ID NO:8).
  • mice were injected with 2- 5x10 ' ' IFU/mouse of the lentiviral construct in 300 ⁇ PBS via either retro-orbital or tail-vein injections.
  • days 9 (Exp. 1) and 14 (Exp. 2) after the injection the mice were sacrificed and total blood, was collected.
  • the cholesterol distribution in plasma lipoproteins fractions was assessed by fast-performance liquid chromatography (FPLC) gel filtration using 250 ⁇ of pooled serum.
  • FPLC fast-performance liquid chromatography
  • mice overexpressing miR-128-1 exhibited several striking phenotypes.
  • Example 6 Effect of miR-128-1 Manipulations on Metabolic Homeostasis in Mice
  • mice on different types of diets either overexpressing miR-128-1 , or subjected to miR-128-1 antisense inhibition.
  • Twenty C57BL/6J mice on regular chow are subjected to tail-vein injection with a miR-128-1 lentivirus, e.g., as described above, while another twenty mice are injected with control (empty vector) vims.
  • control empty vector
  • Similar in vivo experiments are performed with animals fed either a Western-type diet (41% of calories from fat), or a high-fat diet (60% of calories as fat).
  • the animals are weighed every three days. Food intake, and general health and activity, will be monitored daily. After 10 days, blood will be drawn and serum levels of total cholesterol, triglycerides, glucose, and liver enzymes (ALT/AST, for toxicity) will be analyzed, in addition, FPLC analysis of serum lipoproteins (LDL, HDL, and VLDL) will be carried out.
  • mice from each cohort above are divided into two groups, one receiving subcutaneous injection with 5 mg/kg of L A-antisense oligonucleotides

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Abstract
EP12734471.1A 2011-01-14 2012-01-13 Procédés de ciblage du mir-128 en vue de la régulation du métabolisme du cholestérol/des lipides Active EP2663323B1 (fr)

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US9476046B2 (en) 2016-10-25
US9789132B2 (en) 2017-10-17
US20140051746A1 (en) 2014-02-20
WO2012097261A2 (fr) 2012-07-19
US9045749B2 (en) 2015-06-02
US20150232848A1 (en) 2015-08-20

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